14 research outputs found

    Genetic and ultrastructural analysis reveals the key players and initial steps of bacterial magnetosome membrane biogenesis

    Get PDF
    Magnetosomes of magnetotactic bacteria contain well-ordered nanocrystals for magnetic navigation and have recently emerged as the most sophisticated model system to study the formation of membrane bounded organelles in prokaryotes. Magnetosome biosynthesis is thought to begin with the formation of a dedicated compartment, the magnetosome membrane (MM), in which the biosynthesis of a magnetic mineral is strictly controlled. While the biomineralization of magnetosomes and their subsequent assembly into linear chains recently have become increasingly well studied, the molecular mechanisms and early stages involved in MM formation remained poorly understood. In the Alphaproteobacterium Magnetospirillum gryphiswaldense, approximately 30 genes were found to control magnetosome biosynthesis. By cryo-electron tomography of several key mutant strains we identified the gene complement controlling MM formation in this model organism. Whereas the putative magnetosomal iron transporter MamB was most crucial for the process and caused the most severe MM phenotype upon elimination, MamM, MamQ and MamL were also required for the formation of wild-type-like MMs. A subset of seven genes (mamLQBIEMO) combined within a synthetic operon was sufficient to restore the formation of intracellular membranes in the absence of other genes from the key mamAB operon. Tracking of de novo magnetosome membrane formation by genetic induction revealed that magnetosomes originate from unspecific cytoplasmic membrane locations before alignment into coherent chains. Our results indicate that no single factor alone is essential for MM formation, which instead is orchestrated by the cumulative action of several magnetosome proteins

    The genetic architecture of the human cerebral cortex

    Get PDF
    The cerebral cortex underlies our complex cognitive capabilities, yet little is known about the specific genetic loci that influence human cortical structure. To identify genetic variants that affect cortical structure, we conducted a genome-wide association meta-analysis of brain magnetic resonance imaging data from 51,665 individuals. We analyzed the surface area and average thickness of the whole cortex and 34 regions with known functional specializations. We identified 199 significant loci and found significant enrichment for loci influencing total surface area within regulatory elements that are active during prenatal cortical development, supporting the radial unit hypothesis. Loci that affect regional surface area cluster near genes in Wnt signaling pathways, which influence progenitor expansion and areal identity. Variation in cortical structure is genetically correlated with cognitive function, Parkinson's disease, insomnia, depression, neuroticism, and attention deficit hyperactivity disorder

    Testing a global standard for quantifying species recovery and assessing conservation impact.

    Get PDF
    Recognizing the imperative to evaluate species recovery and conservation impact, in 2012 the International Union for Conservation of Nature (IUCN) called for development of a "Green List of Species" (now the IUCN Green Status of Species). A draft Green Status framework for assessing species' progress toward recovery, published in 2018, proposed 2 separate but interlinked components: a standardized method (i.e., measurement against benchmarks of species' viability, functionality, and preimpact distribution) to determine current species recovery status (herein species recovery score) and application of that method to estimate past and potential future impacts of conservation based on 4 metrics (conservation legacy, conservation dependence, conservation gain, and recovery potential). We tested the framework with 181 species representing diverse taxa, life histories, biomes, and IUCN Red List categories (extinction risk). Based on the observed distribution of species' recovery scores, we propose the following species recovery categories: fully recovered, slightly depleted, moderately depleted, largely depleted, critically depleted, extinct in the wild, and indeterminate. Fifty-nine percent of tested species were considered largely or critically depleted. Although there was a negative relationship between extinction risk and species recovery score, variation was considerable. Some species in lower risk categories were assessed as farther from recovery than those at higher risk. This emphasizes that species recovery is conceptually different from extinction risk and reinforces the utility of the IUCN Green Status of Species to more fully understand species conservation status. Although extinction risk did not predict conservation legacy, conservation dependence, or conservation gain, it was positively correlated with recovery potential. Only 1.7% of tested species were categorized as zero across all 4 of these conservation impact metrics, indicating that conservation has, or will, play a role in improving or maintaining species status for the vast majority of these species. Based on our results, we devised an updated assessment framework that introduces the option of using a dynamic baseline to assess future impacts of conservation over the short term to avoid misleading results which were generated in a small number of cases, and redefines short term as 10 years to better align with conservation planning. These changes are reflected in the IUCN Green Status of Species Standard

    Ultrastructural analysis of magnetosome membranes from wild type.

    No full text
    <p>A): Segmented cryo-electron tomogram of cell with selected details from x-y slices of tomographic reconstruction (Box 1–4) and a y-z slice, illustrating information loss by the missing wedge. The outer and cytoplasmic membrane (CM) are depicted in blue, magnetosome membranes (MMs) in yellow, magnetite crystals in red and the magnetosome filament in green. Scale bars in boxes: 100 nm. (B): Segmented cryo-electron tomogram of aerobically cultivated cell that contains MMs (some with small crystal). Full tomogram is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006101#pgen.1006101.s016" target="_blank">S2 Video</a>. (C): Panel with details from x-y slices of tomographic reconstructions of MSR-1 wild type cells, showing MMs that contain magnetite crystals of different sizes. The magnetosome filament is indicated by white arrows. The halo visible around magnetite crystals (red arrow shows examples) is caused by missing wedge effects and might obscure MM identification. (Cii): Section of x-y slice from tomogram showing MM that is continuous with CM and contains a small crystal. Numbers in image represent average value for all measured MM diameters (blue) (n = 289), approximate values for the annulus diameter to the periplasmic space of continuous MMs (green) and approximate values for the length of the protruding neck between the CM and MM (red). Scale bars: 100 nm.</p

    Hypothetical model for magnetosome membrane formation.

    No full text
    <p>The model suggests that magnetosome membrane proteins (colorful shapes) are recruited to certain sites of the cytoplasmic membrane in a hierarchical manner, with the key proteins MamB, MamM, MamQ and MamL (labeled in shades of blue, yellow and red) acting as nucleating factors, which is followed by recruitment of MamI, MamE, MamO and other magnetosome proteins. Since MamB was found most important for magnetosome membrane (MM) formation, it might act as the initial landmark protein to prime complex formation at certain sited within the cytoplasmic membrane. After a critical size and composition of the multi-protein assembly is reached, the formed lipid-protein complex induces rapid invagination to form the magnetosome lumen. Diffusion from the periplasm into this lumen is blocked. Later, several further magnetosome proteins might become recruited into the MM, which eventually becomes detached to form magnetosome vesicles. The absence of MamB strongly inhibits MM formation, while the absence of either MamM, MamQ or MamL might cause a disturbed protein composition, which leads to the formation of aberrant dense magnetosome membrane-like structures (DMMs) that lack magnetite crystals or blocks magnetosome formation at an immature state.</p

    Localization of MamI-GFP in several mutants and complementation/localization assay of mutated MamL-GFP.

    No full text
    <p>(A) Effects of exchange of basic amino acid residues in the C-terminus of MamL, fused to EGFP by an alpha-helical linker. Quantitative analysis of magnetite crystal number/cell (grey) and magnetite crystal sizes (white) of MSR Δ<i>mamL</i> complemented with transposon-integrated P<sub><i>mamDC45</i></sub>-<i>mamL-egfp</i>, P<sub><i>mamDC45</i></sub>-<i>mamL</i><sub>K77Q-R78Q</sub><i>-egfp</i>, P<sub><i>mamDC45</i></sub>-<i>mamL</i><sub>K72Q</sub><i>-egfp</i>, P<sub><i>mamDC45</i></sub>-<i>mamL</i><sub>K63Q-K66Q-K68Q</sub><i>-egfp</i>, P<sub><i>mamDC45</i></sub>-<i>mamL</i><sub>H67Y</sub><i>-egfp</i>, P<sub><i>mamDC45</i></sub>-<i>mamL</i><sub>R64Q-R65Q</sub><i>-egfp</i> and P<sub><i>mamDC45</i></sub>-<i>mamL</i><sub>all neutral</sub><i>-egfp</i>. Box plots are indicating 10<sup>th</sup> and 90<sup>th</sup> percentiles (whiskers), 25<sup>th</sup> and 75<sup>th</sup> percentiles (box), median and outliers. Over 100 cells and 200 magnetosomes where analyzed for each strain. For quantitative analysis of MamL<sub>(mutant)</sub>-EGFP localization (colorful bars in background), fluorescence patterns were grouped into three classes (examples are indicated; chain and aligned patches are visualized as one class). Scale bars: 2 μm. (B) Quantitative analysis of localization of plasmid expressed P<sub><i>mamDC45</i></sub>-<i>mamI</i>-<i>egfp</i> in MSR-1, MSR 1B, Δ<i>mamB</i>, Δ<i>mamM</i>, Δ<i>mamQ</i> and Δ<i>mamL</i>. The localization patterns in individual cells were grouped into four different classes (examples are indicated; boundary of cells are outlined). More than 100 cells where analyzed for each strain. Scale bars: 2 μm.</p

    Induction of <i>mamB</i> and <i>mamB</i>-GFP expression enables <i>de novo</i> magnetosome formation.

    No full text
    <p>(Ai): Progression of growth (OD<sub>565</sub> [circles]) and magnetic response (C<sub>mag</sub> [triangles]) over time after induction of <i>mamB</i> expression with 2 mM IPTG in strain MamB<sub>ind</sub>. IPTG was added at time point 0 (black triangle). TEM micrographs of formaldehyde-fixed cells were utilized to determine the number of magnetite particles per cell (box plots) and average magnetite particle diameter (white boxes) at certain time points. (Aii): Western blot with immune-detection against MamB after SDS-PAGE with whole cell samples from experiment (i) taken at certain time points. The cell density of the samples was normalized based on OD<sub>565</sub>. (Aiii): Examples of TEM micrographs of formaldehyde-fixed cells obtained at different time points of experiment (i). Black arrows indicate the positions of single or multiple magnetite crystals (tiny particles after one hour of induction might stem from background). (B): Details of x-y slices from cryo-electron tomograms acquired with MamB<sub>ind</sub> cells, plunge-frozen at a various time points after induction with 2 mM IPTG in a separate experiment. (i) and (ii): Details from two cells 2 hours post induction. (iii-v): Details from one cell 3 hours post induction. (v-viii): Details from two cells 4 hours post induction. Putative DMMs are indicated by black arrows, the magnetosome filament by white arrows. Scale bars: 100 nm. (C): 24 hours time-lapse live-cell fluorescent microscopy of induced MamB-EGFP<sub>ind</sub> strain. Cells were grown at 30°C on sealed 1% agarose pads containing modified FSM medium and 3 mM IPTG. Fluorescence and corresponding bright field images from various indicated time points after induction are shown. White arrowheads indicate accumulation of fluorescent patches at midcell, while white arrows indicate linear fluorescence signals within cells. Scale bar: 2 μm.</p

    Cryo-electron tomograms of different MSR-1 mutant strains.

    No full text
    <p>Segmented tomograms show representative phenotypes of mutants (compare with <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006101#pgen.1006101.s025" target="_blank">S1 Table</a>). The inner and outer membrane of the cells are depicted in blue, wild type-like magnetosome membranes (MMs) in yellow [black arrow in x-y slices], iron-minerals in red and the magnetosome filament in green [white arrow in x-y slices]. Distinguishable dense magnetosome membrane-like structures (DMMs) are depicted in dark yellow [red arrows in x-y slices], emphasizing the differential appearance in contrast to wild type-like MMs. Full tomograms are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006101#pgen.1006101.s017" target="_blank">S3</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006101#pgen.1006101.s022" target="_blank">S8</a> Videos. (A): Δ<i>mamI</i> cell containing wild type-like MMs that partially enclose mineral particles. (B): Δ<i>mamN</i> cell containing a dense chain of empty and partially magnetite-filled wild type-like MMs. (C): Δ<i>mamL</i> cell with x-y slice detail (Box 1), showing small wild type-like MMs, partially containing crystals, and potential DMMs. (Di): Δ<i>mamB</i> cell displaying some putative isolated DMMs. (Dii): x-y slice detail of another tomogram shows putative DMMs (dashed red arrows) and a “mini-inclusion” structure (blue arrow) occasionally seen also in tomograms of the wild type and several other mutants. Asterisks marks polyhydroxyalkanoate inclusion that also occurred in all other analyzed strains. (Ei): Δ<i>mamQ</i> cell with two x-y slice details (Box 1 and Box 2). Box 1 shows filament-attached DMMs, Box 2 shows putative wild-type like MM. (Eii): x-y slice sections of another tomogram show four putative DMMs of which some appear continuous with the cytoplasmic membrane. Scale bars: 50 nm (F): Δ<i>mamM</i> cell with two x-y slice details (Box 1 and Box 2) showing filament attached DMMs.</p

    Diameters of magnetosome membranes and similar structures from wild type and several mutant strains.

    No full text
    <p>Diameters of magnetosome membranes (MMs) or dense MM-like structures were measured from cryo-electron tomograms. Box plots are indicating 10<sup>th</sup> and 90<sup>th</sup> percentiles (whiskers), 25<sup>th</sup> and 75<sup>th</sup> percentiles (box), median and outliers. The number of measured membranes [n] and analyzed cells [n(cells)] are indicated. The mean value and the standard deviation (SD) of the diameters are given for each strain.</p
    corecore